The rLVS Δ capB/ iglABC vaccine provides potent protection in Fischer rats against inhalational tularemia caused by various virulent Francisella tularensis strains

Abstract

Francisella tularensis is one of the several biothreat agents for which a licensed vaccine is needed. To ensure vaccine protection is achieved across a range of virulent F. tularensis strains, we assembled and characterized a panel of F. tularensis isolates to be utilized as challenge strains. A promising tularemia vaccine candidate is rLVS ΔcapB/iglABC (rLVS), in which the vector is the LVS strain with a deletion in the capB gene and which additionally expresses a fusion protein comprising immunodominant epitopes of proteins IglA, IglB, and IglC. Fischer rats were immunized subcutaneously 1-3 times at 3-week intervals with rLVS at various doses. The rats were exposed to a high dose of aerosolized Type A strain Schu S4 (FRAN244), a Type B strain (FRAN255), or a tick derived Type A strain (FRAN254) and monitored for survival. All rLVS vaccination regimens including a single dose of 10⁷ CFU rLVS provided 100% protection against both Type A strains. Against the Type B strain, two doses of 10⁷ CFU rLVS provided 100% protection, and a single dose of 10⁷ CFU provided 87.5% protection. In contrast, all unvaccinated rats succumbed to aerosol challenge with all of the F. tularensis strains. A robust Th1-biased antibody response was induced in all vaccinated rats against all F. tularensis strains. These results demonstrate that rLVS ΔcapB/iglABC provides potent protection against inhalational challenge with either Type A or Type B F. tularensis strains and should be considered for further analysis as a future tularemia vaccine.

Keywords: Francisella tularensis; LVS (Live Vaccine Strain); aerosol challenge; animal model; rat; tularemia; vaccines.

Figure 1.

Survival data of rats challenged with aerosolized F. tularensis strains for determination of LD₅₀. Groups of Fischer rats (n = 8/group) were challenged by whole body aerosolization with strains FRAN244, FRAN254, or FRAN255 as indicated, and survival monitored. The calculated LD₅₀ values from these experiments are shown in Table 1.

Figure 2.

Efficacy of rLVS against aerosol challenge with F. tularensis (FRAN244) in Fischer rats. (a). Experimental schedule. Fischer rats (n = 7 or 8/group) were immunized subcutaneously (SQ) once with PBS (n = 7/group), 10⁷ CFU LVS (n = 8/group), or 10⁷ CFU rLVS (n = 7/group); twice with 10⁶ CFU rLVS (n = 8/group), 10⁷ CFU rLVS (n = 7/group), or 10⁸ CFU rLVS (n = 7/group) rLVS; or three times with 10⁷ CFU rLVS (n = 8/group), 3 weeks apart; challenged with aerosolized F. tularensis FRAN244 (72 LD₅₀) at Week 9; and monitored for signs of illness, weight change, and death for 3 weeks post challenge, as indicated. (b). Survival post immunization and respiratory challenge. The survival curves are compared by log-rank test for trend, ****, p < 0.0001. (c). All rats were weighed weekly. Weight change post immunization and post respiratory challenge.

Figure 3.

Efficacy of rLVS against aerosol challenge with F. tularensis FRAN254 and FRAN255 in Fischer rats. (a). Experimental schedule. Fischer rats were immunized subcutaneously (SQ) once at Week 0 with PBS (naïve, n = 6 and 7/group for Type A and Type B challenge, respectively), 10⁷ CFU LVS (n = 7 and 8/group for Type A and Type B challenge, respectively), 10⁷ CFU rLVS (n = 8/group for both Type A and Type B challenge) or twice at Week 0 and Week 3 with 10⁸ CFU rLVS (n = 8/group for both Type A and Type B challenge); challenged with aerosolized F. tularensis Type A (FRAN254, 233 LD₅₀) or Type B (FRAN255, 73 LD₅₀) at Week 6; and monitored for signs of illness, weight change, and death for 3 weeks, as indicated. (b). Weight change and survival after vaccination and challenge. Weight changes were compared between any two groups by one-way ANOVA with Tukey’s post comparison’s test. No significant differences were found. The survival curves are compared by log-rank test for trend (Prism 9.2.0). ***, p < 0.001; ****, p < 0.0001.

Figure 4.

Histopathology of lungs from F. tularensis challenged rats. Rats were unvaccinated (naïve) or vaccinated with rLVS, or LVS, as indicated; challenged with FRAN244 (top), FRAN254 (middle), or FRAN255 (bottom); and monitored for survival for 21 days. All naïve rats were necropsied at death or following euthanasia when moribund. Vaccinated rats survived and were euthanized and necropsied at the end of the experiment; however one rLVS (single dose) rat challenged with FRAN255 did succumb). Lungs were examined for histopathology. A representative animal from each group is shown. For the rLVS group, all images shown are from the single dose 10⁷ CFU vaccinated group.

Figure 5.

Total serum IgG antibody in rats pre- and post-challenge (survivors) with Type A F. tularensis FRAN244/Schu S4 strain. Animals were immunized and challenged as described in the legend to Figure 2. Serum IgG antibody and subclasses specific to irradiated FRAN244 (Type A antigen) were assayed. a. Total serum antibody and subclasses in immunized rats prior to challenge with aerosolized F. tularensis Type A FRAN244/Schu S4 strain. For IgG, IgG2a, IgG2b, and IgG2c, all vaccinated groups had significantly greater titers than the PBS group (p<0.01-p<0.001). For IgG1, all vaccinated groups except LVS, rLVS 10⁷ x1 rLVS 10⁷ x2 groups had significantly greater titers than the PBS group (p <0.05 - p <0.001). Values represent median with interquartile range (Q1 and Q3 for the whiskers) of serum antibody for n = 7 or 8 per group. b. Total serum antibody and subclasses in immunized rats surviving challenge with aerosolized F. tularensis Type A strains. Values represent median with interquartile range (Q1 and Q3 for the whiskers) of serum antibody. Notes: in Fischer rats, IgG2b/2c indicates a Th1 type response and IgG1/2a indicates a Th2 type response. Values that are significantly different between two groups are marked with asterisk(s) over an open horizontal line crossing above the two groups. * p <0 .05; ** p < 0.01; and *** p < 0.001. Additional descriptive statistical values are included in Supplemental Tables S2–5.

Figure 6.

Total serum IgG antibody in rats immunized with rLVS vaccines prior to and post challenge with Type A F. tularensis FRAN254 or Type B FRAN255 strain. Animals were immunized and challenged as described in the legend to Figure 3. Sera were collected in immunized rats prior to and after challenge with Type A or Type B strains and assayed for total IgG antibody specific to irradiated FRAN244 (Type A antigen) and FRAN255 (Type B antigen). (a). Prior to challenge; All vaccinated groups in Panel a had significantly greater titers than the PBS group (p <0.01-p <0.0001). (b). Post challenge (survivors only). Values represent median with interquartile range (Q1 and Q3 for the whiskers) of serum antibody titers for n = 6–8/group. * p < 0.05; ** p <0 .01; and *** p < 0.001. Additional descriptive statistical values are included in Supplemental Tables S6,7.